Allergic reactions to house dust mite are an important health problem worldwide, affecting up to 85% of asthmatic children, and are a risk factor for emergency room admission with asthma. Group 1 and 2 mite allergens account for more than 50% of total house dust mite specific IgE reactivity in mite allergic patients. Mite allergens Der p 1 and Der f 1 are cysteine proteases produced by the dust mites Dermatophagoides pteronyssinus and D. farinae, respectively. Proteolytic activity of Der p 1 may enhance IgE antibody production and contribute to lung inflammation in asthma. In contrast, group 2 mite allergens are lipopolysaccharide binding proteins. Der p 2 has been reported to mimic a human structural-homolog that activates the innate immune system through toll like receptors. Despite these important molecular differences between proteolytic group 1 and non-proteolytic group 2, a high IgE prevalence of 83% to allergens from both groups has been observed in mite allergic patients in the US. The main goal of this project is to investigate the antigenic structure of both groups of mite allergens, for the design of immunotherapy. Allergens will be co-crystallized with IgE antibody constructs selected by phage display technology. The key amino acids involved in IgE antibody binding will be identified and modified. The specific aims are: 1) selection of IgE antibody constructs (scFv) from combinatorial libraries made from blood of mite allergic patients using phage display technology; 2) mapping of antigenic determinants on groups 1 and 2 dust mite allergens by X-ray crystallography and analysis of IgE antibody binding epitopes; and 3) site-directed mutagenesis of IgE antibody epitopes for expression of hypoallergenic mutants with T cell reactivity as candidates for immunotherapy. An analysis of the association between mite allergen-specific IgE antibodies from the human repertoire and the epitopes recognized by these IgE antibodies will be performed with the information obtained in the first two aims. Aim #2 will generate experimental data sets of three-dimensional structures of B-cell epitopes that are missing in current databases used for developing tools for B cell epitope prediction. Most importantly, this project will define IgE antibody responses to mite allergens and will provide the structural basis for rational design of hypoallergens. In Aim #3, IgE antibody binding to the epitope mutants will be analyzed by ELISA, multiplex array technology and cell mediator release assays, and T cell reactivity will be evaluated. Mutants will be compared and hypoallergenic forms will be selected for the design of vaccines for immunotherapy of mite allergy.